The proper intracellular distribution of lipids is vital for many cellular functions. Relatively little is known, however, about the means by which cells determine the lipid composition of organelles and regulate intracellular lipid transport. Numerous diseases, including heart disease and Type 2 diabetes, are associated with defects in these processes. The lipid composition of organelles is determined by both modulating the lipid composition of intracellular transport vesicles and nonvesicular lipid transport between organelles. Neither process is well understood. We seek to identify proteins required for nonvesicular lipid trafficking in cells and understand their role in organelle biogenesis and lipid metabolism. Identification of these proteins is critical for understanding organelle biogenesis and how defects in lipid transport contribute to many diseases. To this end, we use a combination of biochemical and genetic approaches in the model organism S. cerevisiae. We are also studying a novel nuclear import pathway used by a transcription factor that regulates lipid metabolism. 1. Two plasma membrane (PM) ABC-transporters mediate raft-regulated nonvesicular sterol transport from the PM to the ER (JBC, in press). We are studying the uptake and intracellular trafficking of exogenous cholesterol and how cells maintain the high sterol content of the PM. We have demonstrated that exogenous sterol can be moved from the PM to the ER by a nonvesicular pathway. This transport is facilitated by either of two ABC-transporters in the PM. This important class of transporters had been implicated previously in sterol efflux from cells and mutations in them cause Tangier?s disease and sitosterolemia. Ours is the first demonstration that they can also mediate intracellular sterol transport. We found that the propensity of a sterol to be moved from the PM to the ER inversely correlates with its ability to associate with sterol-sphingolipid microdomains (rafts) in the PM; rafts are highly enriched in the PM. Finally, we have preliminary evidence, discussed below, that oxysterol-binding proteins may also play a role in the nonvesicular transport of sterol from the PM to the ER. 2. Identification of a RAN-independent, calmodulin-dependent nuclear localization signal (NLS) in the high mobility group (HMG) transcription factor Nhp6ap. We have identified the NLS required for the RAN-independent import of Nhp6ap and demonstrated that it can bind calmodulin, a protein that may play a role in a novel nuclear import pathway. Mutants with calmodulin defects fail to localize a protein containing the Nhp6ap NLS to the nucleus. These mutants do not affect the localization of a reporter with a RAN-dependent NLS. Thus, Nhp6ap is imported into the nucleus by a novel, calmodulin-dependent, RAN-independent nuclear pathway. These results are described in a manuscript that is being prepared. We are isolating mutants with defects in this novel import pathway. 3. Reconstitution of nonvesicular lipid transport from the ER to peroxisomes, mitochondria, and lipid particles. Peroxisomes, mitochondria, and lipid particles all play critical roles in lipid homeostasis and other vital cellular functions. Since there is no known vesicular transport between these organelles and other compartments in the cell, they must acquire lipids by nonvesicular mechanisms. We have reconstituted nonvesicular lipid transport to these compartments in vitro. In all cases, we find that there is rapid, nonvesicular lipid transport between ER-derived vesicles and these organelles. This transport does not require cytosol or ATP, but does require proteins on the ER and the target organelle. This is the first demonstration of nonvesicular lipid transport between the ER and peroxisomes and lipid droplets. These results are described in manuscripts that are being prepared.